Transducin beta-1 subunit

ABSTRACT

The invention provides a human transducin beta-1 subunit (TBS) and polynucleotides which identify and encode TBS. The invention also provides expression vectors, host cells, agonists, antibodies, and antagonists. The invention also provides methods for treating or preventing diseases associated with expression of TBS.

[0001] This application is a divisional application of U.S. applicationSer. No. 09/489,506, filed Jan. 21, 2000, which is a divisionalapplication of U.S. application Ser. No. 08/965,600, filed Nov. 6, 1997,now U.S. Pat. No. 6,077,688, issued Jun. 20, 2000, all of whichapplications and patents are hereby incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a transducin beta-1 subunit and to the use of these sequences in thediagnosis, prevention, and treatment of diseases associated with cellproliferation.

BACKGROUND OF THE INVENTION

[0003] Membrane-bound GTP-binding (G) proteins mediate signaltransduction in a variety of cell systems. Although, the exactmechanisms of G protein action are still under investigation, G proteinsappear to involve effectors located in the plasma membrane as well asother parts of the cell. WD-40 proteins contain a loosely conservedrepeat of approximately 40 amino acids separated by a Trp-Asp dipeptidesequence which may recur several times within each polypeptide. Theconserved core of the WD-40 sequence, which usually ends with the aminoacids Trp-Asp (WD), was first identified in the β-subunit of theheterotrimeric G protein, transducin. Several dozen WD-40 proteins havesince been identified, none are enzymes, and all seem to have regulatoryfunctions (Neer, E. J. et al. (1994) Nature 371:297-300).

[0004] Many of the WD-40 proteins are homologs of β-transducin andfunction in signal transduction pathways within the cytoplasm. WD-40proteins may participate in complex formation, and may interact withother G protein subunits through the WD-40 region. The β-subunit of Gproteins enhances binding of the G_(α)subunit to receptors and assistsin the assembly of the G-protein :receptor complex. The G_(βγ)subunitbinds to and brings the β-adrenergic receptor kinase, β-ARK, to thereceptor (Touhara, K. et al. (1994) J. Biol. Chem. 269: 10217-10220).

[0005] A number of WD repeat proteins are localized to the nucleus andfunction in the repression of transcription. These include Tup1, Hir1,and Met30 in S.cerevisiae; SCON2 in Neurospora crassa; extra sex combsand Groucho in Drosophila; COP1 in Arabidopsis thaliana; and Rec14 inSchizosaccharomyces pombe. These WD-40 proteins turn off a wide varietyof genes, including those involved in segmentation, sex determination,and neurogenesis, and those involved in photomorphogenesis.

[0006] The WD repeat protein, Rec14, is essential for meioticrecombination in S. pombe. Mutations in the Rec14 gene reduce meioticrecombination by as much as a factor of 1000 on chromosomes I and III.The Rec14 gene contains two exons separated by a 53-bp intron. Thespliced transcript encodes a protein of 302 amino acids and contains sixWD repeat motifs common in the G-beta transduction family and inS.cerevisiae Rec103 protein. Rec103 mutations have no detectable effecton mitotic recombination. Based upon phenotypes and amino acid sequencesimilarities, it is possible that R is a functional homolog of S.cerevisiae Rec103(Evens, D. H. et al. (1997) Genetics 146:1253-1264).Autoantibodies occurring spontaneously in certain cancer patients arereagents for identifying cellular proteins, and it is the WD-40 motif inthe beta-transducin proteins which target these antibodies.

[0007] The discovery of a new transducin beta-1 subunit and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, prevention and treatmentof diseases associated with cell proliferation, in particular, cancersand immune response.

SUMMARY OF THE INVENTION

[0008] The invention features a substantially purified polypeptide,transducin beta-1 subunit (TBS), having the amino acid sequence shown inSEQ ID NO:1, or fragments thereof.

[0009] The invention further provides an isolated and substantiallypurified polynucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or fragments thereof and acomposition comprising said polynucleotide sequence. The invention alsoprovides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence encoding the amino acidsequence SEQ ID NO:1, or fragments of said polynucleotide sequence. Theinvention further provides a polynucleotide sequence comprising thecomplement of the polynucleotide sequence encoding the amino acidsequence of SEQ ID NO:1, or fragments or variants of said polynucleotidesequence.

[0010] The invention also provides an isolated and purified sequencecomprising SEQ ID NO.2 or variants thereof. In addition, the inventionprovides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence of SEQ ID NO:2. The inventionalso provides a polynucleotide sequence comprising the complement of SEQID NO:2, or fragments or variants thereof.

[0011] The present invention further provides an expression vectorcontaining at least a fragment of any of the claimed polynucleotidesequences. In yet another aspect, the expression vector containing thepolynucleotide sequence is contained within a host cell.

[0012] The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof,the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding TBS under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.

[0013] The invention also provides a pharmaceutical compositioncomprising a substantially purified TBS having the amino acid sequenceof SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.

[0014] The invention also provides a purified antagonist of thepolypeptide of SEQ ID NO.1. In one aspect the invention provides apurified antibody which binds to a polypeptide comprising the amino acidsequence of SEQ ID NO:1.

[0015] Still further, the invention provides a purified agonist of thepolypeptide of SEQ ID NO:1.

[0016] The invention also provides a method for treating or preventingan immune response disorder comprising administering to a subject inneed of such treatment an effective amount of an antagonist to TBS.

[0017] The invention also provides a method for treating or preventing acancer comprising administering to a subject in need of such treatmentan effective amount of an antagonist to TBS.

[0018] The invention also provides a method for detecting apolynucleotide which encodes TBS in a biological sample comprising thesteps of: a) hybridizing the complement of the polynucleotide sequencewhich encodes SEQ ID NO:1 to nucleic acid material of a biologicalsample, thereby forming a hybridization complex; and b) detecting thehybridization complex, wherein the presence of the complex correlateswith the presence of a polynucleotide encoding TBS in the biologicalsample. In one aspect the nucleic acid material of the biological sampleis amplified by the polymerase chain reaction prior to hybridization.

BRIEF DESCRIPTION OF THE FIGURES

[0019]FIGS. 1A, 1B, 1C and 1D show the amino acid sequence (SEQ IDNO: 1) and nucleic acid sequence (SEQ ID NO:2) of transducin beta-1subunit (TBS). The alignment was produced using MacDNASIS PRO™ software(Hitachi Software Engineering Co. Ltd. San Bruno, Calif.).

[0020]FIG. 2 shows the amino acid sequence alignments among TBS (194046;SEQ ID NO:1) and Rec14 (GI 1079671; SEQ ID NO:3), produced using themultisequence alignment program of DNASTAR™ software (DNASTAR Inc,Madison Wis.).

[0021]FIGS. 3A and 3B show the hydrophobicity plots for TBS (SEQ IDNO: 1) and Rec14 (SEQ ID NO:3), respectively; the positive X axisreflects amino acid position, and the negative Y axis, hydrophobicity(MacDNASIS PRO software).

DESCRIPTION OF THE INVENTION

[0022] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0023] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0024] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0025] Definitions

[0026] TBS, as used herein, refers to the amino acid sequences ofsubstantially purified TBS obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0027] The term “agonist”, as used herein, refers to a molecule which,when bound to TBS, increases or prolongs the duration of the effect ofTBS. Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to and modulate the effect of TBS.

[0028] An “allele” or “allelic sequence”, as used herein, is analternative form of the gene encoding TBS. Alleles may result from atleast one mutation in the nucleic acid sequence and may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given natural or recombinant gene may have none, one, ormany allelic forms. Common mutational changes which give rise to allelesare generally ascribed to natural deletions, additions, or substitutionsof nucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0029] “Altered” nucleic acid sequences encoding TBS as used hereininclude those with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent TBS. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding TBS, andimproper or unexpected hybridization to alleles, with a locus other thanthe normal chromosomal locus for the polynucleotide sequence encodingTBS. The encoded protein may also be “altered” and contain deletions,insertions, or substitutions of amino acid residues which produce asilent change and result in a functionally equivalent TBS. Deliberateamino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the biological orimmunological activity of TBS is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine,glycine and alanine, asparagine and glutamine, serine and threonine, andphenylalanine and tyrosine.

[0030] “Amino acid sequence” as used herein refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof, and tonaturally occurring or synthetic molecules. Fragments of TBS arepreferably about 5 to about 15 amino acids in length and retain thebiological activity or the immunological activity of TBS. Where “aminoacid sequence” is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, amino acid sequence, and liketerms, are not meant to limit the amino acid sequence to the complete,native amino acid sequence associated with the recited protein molecule.

[0031] “Amplification” as used herein refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0032] The term “antagonist” as used herein, refers to a molecule which,when bound to TBS, decreases the amount or the duration of the effect ofthe biological or immunological activity of TBS. Antagonists may includeproteins, nucleic acids, carbohydrates, antibodies or any othermolecules which decrease the effect of TBS.

[0033] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fa, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind TBSpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or oligopeptide used to immunize an animal can be derivedfrom the translation of RNA or synthesized chemically and can beconjugated to a carrier protein, if desired. Commonly used carriers thatare chemically coupled to peptides include bovine serum albumin andthyroglobulin, keyhole limpet hemocyanin. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

[0034] The term “antigenic determinant”, as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0035] The term “antisense”, as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation “negative” is sometimes used in referenceto the antisense strand, and “positive” is sometimes used in referenceto the sense strand.

[0036] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic TBS, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0037] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

[0038] A “composition comprising a given polynucleotide sequence” asused herein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding TBS (SEQ ID NO:1) or fragments thereof (e.g., SEQ ID NO:2 andfragments thereof) may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

[0039] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, has been extendedusing XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5′ and/or the 3′direction and resequenced, or has been assembled from the overlappingsequences of more than one Incyte Clone using a computer program forfragment assembly (e.g., GELVIEW fragment assembly system, GCG, Madison,Wis). Some sequences have been both extended and assembled to producethe consensus sequence.

[0040] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2 by northern analysis is indicativeof the presence of mRNA encoding TBS in a sample and thereby correlateswith expression of the transcript from the polynucleotide encoding theprotein.

[0041] A “deletion”, as used herein, refers to a change in the aminoacid or nucleotide sequence and results in the absence of one or moreamino acid residues or nucleotides.

[0042] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to TBS or theencoded TBS. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

[0043] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

[0044] Human artificial chromosomes (HACs) are linear microchromosomeswhich may contain DNA sequences of 10K to 10M in size and contain all ofthe elements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355).

[0045] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0046] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0047] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

[0048] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

[0049] “Microarray” refers to an array of distinct polynucleotides oroligonucleotides synthesized on a substrate, such as paper, nylon orother type of membrane, filter, chip, glass slide, or any other suitablesolid support.

[0050] The term “modulate”, as used herein, refers to a change in theactivity of TBS. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional or immunological properties of TBS.

[0051] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments thereof,and to DNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand.“Fragments” are those nucleic acid sequences which are greater than 60nucleotides than in length, and most preferably includes fragments thatare at least 100 nucleotides or at least 1000 nucleotides, and at least10,000 nucleotides in length.

[0052] The term “oligonucleotide” refers to a nucleic acid sequence ofat least about 6 nucleotides to about 60 nucleotides, preferably about15 to 30 nucleotides, and more preferably about 20 to 25 nucleotides,which can be used in PCR amplification or a hybridization assay, or amicroarray. As used herein, oligonucleotide is substantially equivalentto the terms “amplimers”,“primers”, “oligomers”, and “probes”, ascommonly defined in the art.

[0053] “Peptide nucleic acid”, PNA as used herein, refers to anantisense molecule or anti-gene agent which comprises an oligonucleotideof at least five nucleotides in length linked to a peptide backbone ofamino acid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

[0054] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from five amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1” encompasses the full-length TBS and fragments thereof.

[0055] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding TBS,or fragments thereof, or TBS itself may comprise a bodily fluid, extractfrom a cell, chromosome, organelle, or membrane isolated from a cell, acell, genomic DNA, RNA, or cDNA(in solution or bound to a solid support,a tissue, a tissue print, and the like.

[0056] The terms “specific binding” or “specifically binding”, as usedherein, refers to that interaction between a protein or peptide and anagonist, an antibody and an antagonist. The interaction is dependentupon the presence of a particular structure (i.e., the antigenicdeterminant or epitope) of the protein recognized by the bindingmolecule. For example, if an antibody is specific for epitope “A”, thepresence of a protein containing epitope A (or free, unlabeled A) in areaction containing labeled “A” and the antibody will reduce the amountof labeled A bound to the antibody.

[0057] The terms “stringent conditions” or “stringency”, as used herein,refer to the conditions for hybridization as defined by the nucleicacid, salt, and temperature. These conditions are well known in the artand may be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

[0058] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0059] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0060] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0061] A “variant” of TBS, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Analogousminor variations may also include amino acid deletions or insertions, orboth. Guidance in determining which amino acid residues may besubstituted, inserted, or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNASTAR software.

[0062] The Invention

[0063] The invention is based on the discovery of a new human transducinbeta-1 subunit (hereinafter referred to as “TBS”), the polynucleotidesencoding TBS, and the use of these compositions for the diagnosis,prevention, or treatment of cancers and immune response.

[0064] Nucleic acids encoding the TBS were first identified in IncyteClone 194046 from the kidney cDNA library (KIDNNOT02) using a computersearch for amino acid sequence alignments. A consensus sequence, SEQ IDNO:2, was derived from the following overlapping and/or extended nucleicacid sequences: Incyte Clones 194046 (KIDNNOT02), 2550636 (LUNGTUT06),104214 (BMARNOT02), and 1857141 (PROSNOT18).

[0065] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A,1B, and 1C. TBS is 305 amino acids in length and has six potentialcasein kinase II phosphorylation sites at residues S₃₉, S₈₂, T₂₀₇, S₂₅₀,T₂₆₉, and S₂₉₁; five potential protein kinase C phosphorylation sites atresidues T₂₆, S₁₃₇, T₁₇₉, S₂₅₂ , and S_(255;) and one potentialbeta-transduction family WD repeats signature site at residue A₇₉. Asshown in FIG. 2, TBS has chemical and structural homology with Rec14 (GI1079671; SEQ ID NO:3). In particular, TBS and Rec14 share 32% identity.As illustrated by FIGS. 3A and 3B, TBS and Rec14 have rather similarhydrophobicity plots. Northern analysis shows the expression of thissequence in various libraries, at least 58% of which are immortalized orcancerous, at least 20% of which involve call proliferation, and atleast 18% of which involve immune response.

[0066] The invention also encompasses TBS variants. A preferred TBSvariant is one having at least 80% and, more preferably at least 90%amino acid sequence identity to the TBS amino acid sequence (SEQ IDNO: 1) and retaining at least one biological, immunological, or otherfunctional characteristic or activity of TBS. A most preferred TBSvariant is one having at least 95% amino acid sequence identity to SEQID NO:1.

[0067] The invention also encompasses polynucleotides which encode TBS.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of TBS can be used to produce recombinant molecules whichexpress TBS. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2, asshown in FIGS. 1A, 1B, and 1C.

[0068] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding TBS, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring TBS, and all such variations are to beconsidered as being specifically disclosed.

[0069] Although nucleotide sequences which encode TBS and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring TBS under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding TBS or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding TBS and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0070] The invention also encompasses production of DNA sequences, orfragments thereof, which encode TBS and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art. Moreover,synthetic chemistry may be used to introduce mutations into a sequenceencoding TBS or any fragment thereof.

[0071] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO:2, under various conditions ofstringency as taught in Wahl, G. M. and S. L. Berger (1987; MethodsEnzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.152:507-511).

[0072] Methods for DNA sequencing which are well known and generallyavailable in the art and may be used to practice any of the embodimentsof the invention. The methods may employ such enzymes as the Klenowfragment of DNA polymerase I, Sequenase® (US Biochemical Corp,Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7polymerase (Amersham, Chicago, Ill.), or combinations of polymerases andproofreading exonucleases such as those found in the ELONGASEamplification system marketed by Gibco/BRL (Gaithersburg, Md.).Preferably, the process is automated with machines such as the MICROLAB2200(Hamilton, Reno, Nev.), Peltier PTC200 thermal cycler (M J Research,Watertown, Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers(Perkin Elmer).

[0073] The nucleic acid sequences encoding TBS may be extended utilizinga partial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

[0074] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 primer analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

[0075] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

[0076] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PromoterFinder™libraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junction screening for full-length cDNAs, it is preferableto use libraries that have been size-selected to include larger cDNAs.Also, random-primed libraries are preferable, in that they will containmore sequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5′ non-transcribedregulatory regions.

[0077] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled devise camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. Genotyper™ andSequence Navigator™, Perkin Elmer) and the entire process from loadingof samples to computer analysis and electronic data display may becomputer controlled. Capillary electrophoresis is especially preferablefor the sequencing of small pieces of DNA which might be present inlimited amounts in a particular sample.

[0078] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode TBS may be used in recombinant DNAmolecules to direct expression of TBS, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressTBS.

[0079] As will be understood by those of skill in the art, it may beadvantageous to produce TBS-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

[0080] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterTBS encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

[0081] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding TBS may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of TBS activity, it may be useful toencode a chimeric TBS protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the TBS encoding sequence and theheterologous protein sequence, so that TBS may be cleaved and purifiedaway from the heterologous moiety.

[0082] In another embodiment, sequences encoding TBS may be synthesized,in whole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 7:215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 7:225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of TBS, or a fragment thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 43 1Apeptide synthesizer (Perkin Elmer).

[0083] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of TBS, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0084] In order to express a biologically active TBS, the nucleotidesequences encoding TBS or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0085] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding TBSand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0086] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding TBS. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

[0087] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may be used.The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding TBS,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0088] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for TBS. For example, whenlarge quantities of TBS are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the sequence encoding TBS may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0089] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0090] In cases where plant expression vectors are used, the expressionof sequences encoding TBS may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology(1992) McGraw Hill, New York,N.Y.; pp. 191-196.

[0091] An insect system may also be used to express TBS. For example, inone such system, Autographa califomica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding TBSmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of TBS will render the polyhedrin gene inactive andproduce recombinant virus lacking coat protein. The recombinant virusesmay then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which TBS may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0092] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding TBS may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing TBS in infected host cells (Logan, J. andShenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0093] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

[0094] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding TBS. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding TBS, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0095] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

[0096] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress TBS may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0097] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418(Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0098] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding TBS isinserted within a marker gene sequence, transformed cells containingsequences encoding TBS can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding TBS under the control of a single promoter. Expressionof the marker gene in response to induction or selection usuallyindicates expression of the tandem gene as well.

[0099] Alternatively, host cells which contain the nucleic acid sequenceencoding TBS and express TBS may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0100] The presence of polynucleotide sequences encoding TBS can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding TBS.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding TBS todetect transformants containing DNA or RNA encoding TBS.

[0101] A variety of protocols for detecting and measuring the expressionof TBS, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson TBS is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0102] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding TBSinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding TBS, or any fragments thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp. (Cleveland,Ohio). Suitable reporter molecules or labels, which may be used for easeof detection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

[0103] Host cells transformed with nucleotide sequences encoding TBS maybe cultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeTBS may be designed to contain signal sequences which direct secretionof TBS through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding TBS to nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and TBS may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingTBS and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281), while the enterokinase cleavage site provides ameans for purifying TBS from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

[0104] In addition to recombinant production, fragments of TBS may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431Apeptide synthesizer (Perkin Elmer). Various fragments of TBS may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

[0105] Therapeutics

[0106] Chemical and structural homology exists between TBS and Rec14from Schizosaccharomyces pombe(GI 1079671). Northern analysis shows thatthe expression of TBS is associated with cell proliferation, and, inparticular, with the presence of cancers, and immune response disorders.

[0107] Therefore, in one embodiment, an antagonist of TBS may beadministered to a subject to treat or prevent an immune responsedisorders of any type and, in particular, that which is associated witha particular disorder. Such disorders include, but are not limited to,AIDS, Addison's disease, adult respiratory distress syndrome, allergies,anenia, asthma, atherosclerosis, bronchitis, cholecystitis, Crohn'sdisease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,glomerulonephritis, gout, Graves'disease, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjogren's syndrome, and autoimmune thyroiditis;complications of cancer, hemodialysis, and extracorporeal circulation;viral, bacterial, fungal, parasitic, protozoal, and helminthicinfections; and trauma. In one aspect, an antibody which specificallybinds TBS may be used directly as an antagonist or indirectly as atargeting or delivery mechanism for bringing a pharmaceutical agent tocells or tissues which express TBS.

[0108] In another embodiment, a vector expressing the complement of thepolynucleotide encoding TBS may be administered to a subject to treat orprevent an immune response as associated with, but not limited to, thedisorders described above.

[0109] In another embodiment, an antagonist of TBS or a fragment orderivative thereof may be administered to a subject to treat or preventa cancer. Such cancers include, but are not limited to, adenocarcinoma,leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma,and, in particular, cancers of the adrenal gland, bladder, bone, bonemarrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinaltract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid,penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid,and uterus. In one aspect, an antibody which specifically binds TBS maybe used directly as an antagonist or indirectly as a targeting ordelivery mechanism for bringing a pharmaceutical agent to cells ortissues which express TBS.

[0110] In another embodiment, a vector capable of expressing thecomplement of the polynucleotide encoding TBS may be administered to asubject to treat or prevent a cancer including, but not limited to,those cancers listed above.

[0111] In other embodiments, any of the proteins, antagonists,antibodies, agonists, complementary sequences or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0112] An antagonist of TBS may be produced using methods which aregenerally known in the art. In particular, purified TBS may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind TBS.

[0113] Antibodies to TBS may be generated using methods that are wellknown in the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0114] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith TBS or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0115] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to TBS have an amino acid sequence consistingof at least five amino acids and more preferably at least 10 aminoacids. It is also preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of TBS amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0116] Monoclonal antibodies to TBS may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0117] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceTBS-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0118] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0119] Antibody fragments which contain specific binding sites for TBSmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0120] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between TBS and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering TBS epitopes is preferred, but a competitive bindingassay may also be employed (Maddox, supra).

[0121] In another embodiment of the invention, the polynucleotidesencoding TBS, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding TBS may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding TBS. Thus, complementary molecules or fragments may be used tomodulate TBS activity, or to achieve regulation of gene function. Suchtechnology is now well known in the art, and sense or antisenseoligonucleotides or larger fragments, can be designed from variouslocations along the coding or control regions of sequences encoding TBS.

[0122] Expression vectors derived from retro viruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encoding TBS.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra).

[0123] Genes encoding TBS can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes TBS. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0124] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′ or regulatory regions of the geneencoding TBS (signal sequence, promoters, enhancers, and introns).Oligonucleotides derived from the transcription initiation site, e.g.,between positions 31 10 and 30 10 from the start site, are preferred.Similarly, inhibition can be achieved using “triple helix” base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0125] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding TBS.

[0126] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0127] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding TBS. Such DNA sequences may be incorporated into a wide varietyof vectors with suitable RNA polymerase promoters such as T7 or SP6.Alternatively, these cDNA constructs that synthesize complementary RNAconstitutively or inducibly can be introduced into cell lines, cells, ortissues.

[0128] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0129] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposome injectionsor polycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

[0130] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0131] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of TBS,antibodies to TBS, mimetics, agonists, antagonists, or inhibitors ofTBS. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0132] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0133] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(MaackPublishing Co., Easton, Pa.).

[0134] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0135] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0136] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0137] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0138] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks'solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Non-lipid polycationic amino polymers may also be used for delivery.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0139] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0140] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0141] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0142] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of TBS, such labeling wouldinclude amount, frequency, and method of administration.

[0143] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0144] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0145] A therapeutically effective dose refers to that amount of activeingredient, for example TBS or fragments thereof, antibodies of TBS,agonists, antagonists or inhibitors of TBS, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, which can be expressed as the LD₅₀/ED₅₀ ratio.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0146] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0147] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0148] Diagnostics

[0149] In another embodiment, antibodies which specifically bind TBS maybe used for the diagnosis of conditions or diseases characterized byexpression of TBS, or in assays to monitor patients being treated withTBS, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for TBS includemethods which utilize the antibody and a label to detect TBS in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0150] A variety of protocols including ELISA, RIA, and FACS formeasuring TBS are known in the art and provide a basis for diagnosingaltered or abnormal levels of TBS expression. Normal or standard valuesfor TBS expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to TBS under conditions suitable for complex formation. Theamount of standard complex formation may be quantified by variousmethods, but preferably by photometric, means. Quantities of TBSexpressed in subject, control and disease, samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0151] In another embodiment of the invention, the polynucleotidesencoding TBS may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof TBS may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression of TBS,and to monitor regulation of TBS levels during therapeutic intervention.

[0152] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding TBS or closely related molecules, may be used to identifynucleic acid sequences which encode TBS. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding TBS, alleles, or related sequences.

[0153] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the TBS encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring TBS.

[0154] Means for producing specific hybridization probes for DNAsencoding TBS include the cloning of nucleic acid sequences encoding TBSor TBS derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

[0155] Polynucleotide sequences encoding TBS may be used for thediagnosis of conditions or disorders which are associated withexpression of TBS.

[0156] Examples of such conditions or disorders include cancers such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; and immune responsedisorders such as AIDS, Addison's disease, adult respiratory distresssyndrome, allergies, anemia, asthma, atherosclerosis, bronchitis,cholecystitis, Crohn's disease, ulcerative colitis, atopic dermatitis,dermatomyositis, diabetes mellitus, emphysema, erythema nodosum,atrophic gastritis, glomerulonephritis, gout, Graves' disease,hypereosinophilia, irritable bowel syndrome, lupus erythematosus,multiple sclerosis, myasthenia gravis, myocardial or pericardialinflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis,rheumatoid arthritis, scleroderma, Sjogren's syndrome, and autoimmunethyroiditis; complications of cancer, hemodialysis, and extracorporealcirculation; viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections; and trauma.

[0157] The polynucleotide sequences encoding TBS may be used in Southernor northern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dipstick, pIN, ELISA assays or microarraysutilizing fluids or tissues from patient biopsies to detect altered TBSexpression. Such qualitative or quantitative methods are well known inthe art.

[0158] In a particular aspect, the nucleotide sequences encoding TBS maybe useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequencesencoding TBS may be labeled by standard methods, and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantitated and compared with astandard value. If the amount of signal in the biopsied or extractedsample is significantly altered from that of a comparable controlsample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding TBS in the sample indicates the presenceof the associated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0159] In order to provide a basis for the diagnosis of diseaseassociated with expression of TBS, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes TBS, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0160] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0161] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0162] Additional diagnostic uses for oligonucleotides designed from thesequences encoding TBS may involve the use of PCR. Such oligomers may bechemically synthesized, generated enzymatically, or produced in vitro.Oligomers will preferably consist of two nucleotide sequences, one withsense orientation (5′→3′) and another with antisense (3′←5′), employedunder optimized conditions for identification of a specific gene orcondition. The same two oligomers, nested sets of oligomers, or even adegenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

[0163] Methods which may also be used to quantitate the expression ofTBS include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and standard curves onto which theexperimental results are interpolated (Melby, P. C. et al. (1993) J.Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.212:229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

[0164] In further embodiments, an oligonucleotide derived from any ofthe polynucleotide sequences described herein may be used as a target ina microarray. The microarray can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information will be useful in determining gene function,understanding the genetic basis of disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents (Heller, R.et al. (1997) Proc. Natl. Acad. Sci. 94:2150-55).

[0165] In one embodiment, the microarray is prepared and used accordingto the methods described in PCT application WO95/11995(Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference.

[0166] The microarray is preferably composed of a large number ofunique, single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably 15-30 nucleotides in length, and most preferablyabout 20-25 nucleotides in length. For a certain type of microarray, itmay be preferable to use oligonucleotides which are only 7-10nucleotides in length. The microarray may contain oligonucleotides whichcover the known 5′, or 3′, sequence, sequential oligonucleotides whichcover the full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of the sequence is knownor that are specific to one or more unidentified cDNAs which are commonto a particular cell type, developmental or disease state.

[0167] In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5′ or more preferably at the 3′ end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In certain situations it may beappropriate to use pairs of oligonucleotides on a microarray. The“pairs” will be identical, except for one nucleotide which preferably islocated in the center of the sequence. The second oligonucleotide in thepair (mismatched by one) serves as a control. The number ofoligonucleotide pairs may range from two to one million. The oligomersare synthesized at designated areas on a substrate using alight-directed chemical process. The substrate may be paper, nylon orother type of membrane, filter, chip, glass slide or any other suitablesolid support.

[0168] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entiretyby reference. In another aspect, a “gridded” array analogous to a dot(or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0169] In order to conduct sample analysis using a microarray, the RNAor DNA from a biological sample is made into hybridization probes. TheiTRNA is isolated, and cDNA is produced and used as a template to makeantisense RNA (aRNA). The aRNA is amplified in the presence offluorescent nucleotides, and labeled probes are incubated with themicroarray so that the probe sequences hybridize to complementaryoligonucleotides of the microarray. Incubation conditions are adjustedso that hybridization occurs with precise complementary matches or withvarious degrees of less complementarity. After removal of nonhybridizedprobes, a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies on the sequences, mutations, variants, or polymorphisms amongsamples.

[0170] In another embodiment of the invention, the nucleic acidsequences which encode TBS may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome, to aspecific region of a chromosome or to artificial chromosomeconstructions, such as human artificial chromosomes (HACs), yeastartificial chromosomes (YACs), bacterial artificial chromosomes (BACs),bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0171] Fluorescent in situ hybridization (FISH as described in Verma etal. (1988) Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York, N.Y.) may be correlated with other physical chromosomemapping techniques and genetic map data. Examples of genetic map datacan be found in various scientific journals or at Online MendelianInheritance in Man (OMIM). Correlation between the location of the geneencoding TBS on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

[0172] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, ataxiatelangiectasia to11q22-23(Gatti, R. A. et al. (1988) Nature 336:577-580), any sequencesmapping to that area may represent associated or regulatory genes forfurther investigation. The nucleotide sequence of the subject inventionmay also be used to detect differences in the chromosomal location dueto translocation, inversion, etc. among normal, carrier, or affectedindividuals.

[0173] In another embodiment of the invention, TBS, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, between TBSand the agent being tested, may be measured.

[0174] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to TBS large numbersof different small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with TBS, or fragments thereof, and washed. Bound TBS is thendetected by methods well known in the art. Purified TBS can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

[0175] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding TBSspecifically compete with a test compound for binding TBS. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with TBS.

[0176] In additional embodiments, the nucleotide sequences which encodeTBS may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0177] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0178] I KIDNNOT02 cDNA Library Construction

[0179] The KIDNNOT02 cDNA library was constructed from a kidney obtainedfrom the Keystone Skin Bank, International Institute for the Advancementof Medicine (Exton, Pa.). Kidney tissue from a 64 year old Caucasianfemale (Lot HEF698) was flash frozen, ground in a mortar and pestle, andlysed immediately in buffer containing guanidinium isothiocyanate. Lysiswas followed by several phenol chloroform extractions and ethanolprecipitation. Poly A+RNA was isolated using biotinylated oligo d(T)primer and streptavidin coupled to a paramagnetic particle (PromegaCorp, Madison Wis.) and sent to Stratagene.

[0180] Stratagene prepared the cDNA library using oligo d(T) priming.Synthetic adapter oligonucleotides were ligated onto the cDNA molecules,which were then inserted into the Uni ZAP-vector system (Stratagene).The quality of the cDNA library was screened using DNA probes, and thePBLUESCRIPT phagemid (Stratagene) was excised. Subsequently, libraryphage particles were infected into E. coli host strain XL1-Blue(Stratagene). Alternative unidirectional vectors might include, but arenot limited to, pcDNAI (Invitrogen, San Diego, Calif.) and pSHlox-1(Novagen, Madison Wis.).

[0181] II Isolation and Sequencing of cDNA Clones

[0182] The phagemid forms of individual cDNA clones were obtained by thein vivo excision process, in which the host bacterial strain wascoinfected with both the lambda library phage and an f1 helper phage.Polypeptides derived from both the library-containing phage and thehelper phage nicked the lambda DNA, initiated new DNA synthesis fromdefined sequences on the lambda target DNA, and created a smaller,single stranded circular phagemid DNA molecule containing all DNAsequences of the PBLUESCRIPT plasmid and the cDNA insert. The phagemidDNA was secreted from the cells and purified and was, then used tore-infect fresh host cells. Double stranded phagemid DNA was producedand selected on medium containing ampicillin.

[0183] Phagemid DNA was purified using the Magic Minipreps™ DNAPurification System (catalogue #A7100. Promega Corp., Madison, Wis.).Alternatively, phagemid DNA was purified using the QIAwell-8, QIAwellPLUS, and QIAwell ULTRA DNA Purification System (QIAGEN). The DNA waseluted from the purification resin already prepared for DNA sequencingand other analytical manipulations.

[0184] The cDNAs were sequenced by the method of Sanger F. and A. R.Coulson (1975; J. Mol. Biol. 94:441f), using a MICROLAB 2200(Hamilton,Reno Nev.) in combination with four Peltier PTC200 thermal cyclers (MJResearch, Watertown Mass.) and Applied Biosystems 377 or 373 DNAsequencing systems (Perkin Elmer), and reading frame was determined.

[0185] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0186] The nucleotide sequences of the Sequence Listing or the aminoacid sequences deduced from them were used as query sequences againstdatabases such as GenBank, SwissProt, BLOCKS, and Pima II. Thesedatabases, which contain previously identified and annotated sequences,were searched for regions of homology (similarity) using BLAST (BasicLocal Alignment Search Tool) (Altschul S. F. (1993) J. Mol. Evol.36:290-300; Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-10).

[0187] BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal or plant) origin. Otheralgorithms, such as the one described in Smith R. F. and T. F. Smith(1992 Protein Engineering 5:35-51), incorporated herein by reference,can be used when dealing with primary sequence patterns and secondarystructure gap penalties. As disclosed in this application, the sequenceshave lengths of at least 49 nucleotides, and no more than 12% uncalledbases (where N is recorded rather than A, C, G, or T).

[0188] The BLAST approach, as detailed in Karlin and Altschul (1993;Proc. Natl. Acad. Sci. 90:5873-7) and incorporated herein by reference,searches matches between a query sequence and a database sequence, toevaluate the statistical significance of any matches found, and toreport only those matches which satisfy the user-selected threshold ofsignificance. In this application, threshold was set at 10-25 fornucleotides and 10-14 for peptides.

[0189] Incyte nucleotide sequence were searched against the GenBankdatabases for primate (pri), rodent (rod), and mammalian (mam)sequences, and deduced amino acid sequences from the same clones aresearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp) and eukaryote (eukp), for homology.

[0190] IV Northern Analysis

[0191] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involving the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0192] Analogous computer techniques using BLAST (Altschul, S. F. (1993)J. Mol. Evol. 36:290-300; Altschul, S. F. et al. (1990) J. Mol. Evol.215:403-410) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0193] The basis of the search is the product score which is defined as:

[0194] % sequence identity×% maximum BLAST score 100

[0195] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show'product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0196] The results of northern analysis are reported as a list oflibraries in which the transcript encoding TBS occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0197] V Extension of TBS Encoding Polynucleotides

[0198] The nucleic acid sequence of the Incyte Clone 194046 was used todesign oligonucleotide primers for extending a partial nucleotidesequence to full length. One primer was synthesized to initiateextension in the antisense direction, and the other was synthesized toextend sequence in the sense direction. Primers were used to facilitatethe extension of the known sequence “outward” generating ampliconscontaining new, unknown nucleotide sequence for the region of interest.The initial primers were designed from the cDNA using OLIGO 4.06software (National Biosciences), or another appropriate program, to beabout 22 to about 30 nucleotides in length, to have a GC content of 50%or more, and to anneal to the target sequence at temperatures of about68° to about 72° C. Any stretch of nucleotides which would result inhairpin structures and primer-primer dimerizations was avoided.

[0199] Selected human cDNA libraries (Gibco/BRL) were used to extend thesequence If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0200] High fidelity amplification was obtained by following theinstructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing theenzyme and reaction mix. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR wasperformed using the Peltier PTC200 thermal cycler (M J Research,Watertown, Mass.) and the following parameters:  Step 1 94° C. for 1 min(initial denaturation)  Step 2 65° C. for 1 min  Step 3 68° C. for 6 min Step 4 94° C. for 15 sec  Step 5 65° C. for 1 min  Step 6 68° C. for 7min  Step 7 Repeat step 4-6 for 15 additional cycles  Step 8 94° C. for15 sec  Step 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11Repeat step 8-10 for 12 cycles Step 12 72° C. for 8 min Step 13  4° C.(and holding)

[0201] A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were excised from the gel,purified using QIAQuick™ (QIAGEN Inc., Chatsworth, Calif.), and trimmedof overhangs using Klenow enzyme to facilitate religation and cloning.

[0202] After ethanol precipitation, the products were redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2×Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2×Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

[0203] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionwere added to each well. Amplification was performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7  4° C. (andholding)

[0204] Aliquots of the PCR reactions were run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products werecompared to the original partial cDNAs, and appropriate clones wereselected, ligated into plasmid, and sequenced.

[0205] In like manner, the nucleotide sequence of SEQ ID NO:2 is used toobtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for 5′ extension, and an appropriate genomiclibrary.

[0206] VI Labeling and Use of Individual Hybridization Probes

[0207] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences), labeled by combining50 pmol of each oligomer and 250 μCi of [γ-³²P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN®, Boston, Mass.).The labeled oligonucleotides are substantially purified with SephadexG-25 superfine resin column (Pharmacia & Upjohn). A aliquot containing10⁷ counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1,or Pvu II; DuPont NEN®).

[0208] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots, or the blots areexposed in a Phosphorlmager cassette (Molecular Dynamics, Sunnyvale,Calif.), hybridization patterns are compared visually.

[0209] VII Microarrays

[0210] To produce oligonucleotides for a microarray, the nucleotidesequence described herein is examined using a computer algorithm whichstarts at the 3′ end of the nucleotide sequence. The algorithmidentifies oligomers of defined length that are unique to the gene, havea GC content within a range suitable for hybridization, and lackpredicted secondary structure that would interfere with hybridization.The algorithm identifies 20 sequence-specific oligonucleotides of 20nucleotides in length (20-mers). A matched set of oligonucleotides iscreated in which one nucleotide in the center of each sequence isaltered. This process is repeated for each gene in the microarray, anddouble sets of twenty 20 mers are synthesized and arranged on thesurface of the silicon chip using a light-directed chemical process(Chee, M. et al., PCT/WO95/11995, incorporated herein by reference).

[0211] In the alternative, a chemical coupling procedure and an ink jetdevice are used to synthesize oligomers on the surface of a substrate(Baldeschweiler, J. D. et al., PCT/WO95/25116, incorporated herein byreference). In another alternative, a “gridded” array analogous to a dot(or slot) blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available materials and machines and containgrids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.After hybridization, the microarray is washed to remove nonhybridizedprobes, and a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the micro-array.

[0212] VIII Complementary Polynucleotides

[0213] Sequence complementary to the TBS-encoding sequence, or any partthereof, is used to decrease or inhibit expression of naturallyoccurring TBS. Although use of oligonucleotides comprising from about 15to about 30 base-pairs is described, essentially the same procedure isused with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using Oligo 4.06 software and the codingsequence of TBS, SEQ ID NO: 1. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the TBS-encoding transcript.

[0214] IX Expression of TBS

[0215] Expression of TBS is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express TBS in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0216] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of TBS into the bacterial growth media which can be useddirectly in the following assay for activity.

[0217] X Demonstration of TBS Activity

[0218] TBS can be expressed in a mammalian cell line such as 293T bytransfecting with an eukaryotic expression vector encoding TBS.Eukaryotic expression vectors are commercially available, and thetechniques to introduce them into cells are well known to those skilledin the art. A small amount of a second plasmid, which expresses any oneof a number of reporter genes such as β-galactosidase, is co-transformedinto the cells in order to allow rapid identification of those cellswhich have taken up and expressed the foreign DNA. The cells arecultured in a defined synthetic medium with concentrations of GTP for atleast 48 hours after transformation to allow expression and accumulationof TBS and β-galactosidase.

[0219] Transformed cells expressing β-galactosidase are stained bluewhen a suitable colorimetric substrate is added to the culture mediaunder conditions that are well known in the art. Increasingconcentrations of GTP induces increasing numbers of reporter genepositive cells (Ren, M. et al. (1996) Proc. Natl. Acad. Sci. 93:5151-5155). GTP-treated cells which were not transformed with the TBSexpression vector are used as controls, as are TBS transfected cellscultured without supplemental GTP.

[0220] XI Production of TBS Specific Antibodies

[0221] TBS that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Selection of appropriateepitopes, such as those near the C-terminus or in hydrophilic regions,is described by Ausubel et al. (supra), and others.

[0222] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems 431A peptide synthesizer usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio iodinated, goat anti-rabbitIgG.

[0223] XII Purification of Naturally Occurring TBS Using SpecificAntibodies

[0224] Naturally occurring or recombinant TBS is substantially purifiedby immunoaffinity chromatography using antibodies specific for TBS. Animmunoaffinity column is constructed by covalently coupling TBS antibodyto an activated chromatographic resin, such as CNBr-activated Sepharose(Pharmacia & Upjohn). After the coupling, the resin is blocked andwashed according to the manufacturer's instructions.

[0225] Media containing TBS is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of TBS (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/TBS binding (e.g., a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and TBS is collected.

[0226] XIII Identification of Molecules Which Interact with TBS

[0227] TBS or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled TBS, washed and any wells withlabeled TBS complex are assayed. Data obtained using differentconcentrations of TBS are used to calculate values for the number,affinity, and association of TBS with the candidate molecules.

[0228] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 3 1 305 PRT Homo sapiens misc_feature Incyte ID No 194046 1 Met ThrAsn Gln Tyr Gly Ile Leu Phe Lys Gln Glu Gln Ala His 1 5 10 15 Asp AspAla Ile Trp Ser Val Ala Trp Gly Thr Asn Lys Lys Glu 20 25 30 Asn Ser GluThr Val Val Thr Gly Ser Leu Asp Asp Leu Val Lys 35 40 45 Val Trp Lys TrpArg Asp Glu Arg Leu Asp Leu Gln Trp Ser Leu 50 55 60 Glu Gly His Gln LeuGly Val Val Ser Val Asp Ile Ser His Thr 65 70 75 Leu Pro Ile Ala Ala SerSer Ser Leu Asp Ala His Ile Arg Leu 80 85 90 Trp Asp Leu Glu Asn Gly LysGln Ile Lys Ser Ile Asp Ala Gly 95 100 105 Pro Val Asp Ala Trp Thr LeuAla Phe Ser Pro Asp Ser Gln Tyr 110 115 120 Leu Ala Thr Gly Thr His ValGly Lys Val Asn Ile Phe Gly Val 125 130 135 Glu Ser Gly Lys Lys Glu TyrSer Leu Asp Thr Arg Gly Lys Phe 140 145 150 Ile Leu Ser Ile Ala Tyr SerPro Asp Gly Lys Tyr Leu Ala Ser 155 160 165 Gly Ala Ile Asp Gly Ile IleAsn Ile Phe Asp Ile Ala Thr Gly 170 175 180 Lys Leu Leu His Thr Leu GluGly His Ala Met Pro Ile Arg Ser 185 190 195 Leu Thr Phe Ser Pro Asp SerGln Leu Leu Val Thr Ala Ser Asp 200 205 210 Asp Gly Tyr Ile Lys Ile TyrAsp Val Gln His Ala Asn Leu Ala 215 220 225 Gly Thr Leu Ser Gly His AlaSer Trp Val Leu Asn Val Ala Phe 230 235 240 Cys Pro Asp Asp Thr His PheVal Ser Ser Ser Ser Asp Lys Ser 245 250 255 Val Lys Val Trp Asp Val GlyThr Arg Thr Cys Val His Thr Phe 260 265 270 Phe Asp His Gln Asp Gln ValTrp Gly Val Lys Tyr Asn Gly Asn 275 280 285 Gly Ser Lys Ile Val Ser ValGly Asp Asp Gln Glu Ile His Ile 290 295 300 Tyr Asp Cys Pro Ile 305 21221 DNA Homo sapiens misc_feature Incyte ID No 194046 2 cgcggacccggcttccgacg tgcagcctgg cagtgcagtg agctgtctgg ccttttgtcc 60 ttgatccttggttaaggaaa tgaccaacca gtacggtatt ctcttcaaac aagagcaagc 120 ccatgatgatgccatttggt cagttgcttg ggggacaaac aagaaggaaa actctgagac 180 agtggtcacaggctccctag atgacctggt gaaggtctgg aaatggcgtg atgagaggct 240 ggacctacagtggagtctgg agggacatca gctgggagtg gtgtctgtgg acatcagcca 300 caccctgcccattgctgcat ccagctctct tgatgctcat attcgtcttt gggacttgga 360 aaatggcaaacagataaagt ccatagatgc aggacctgtg gatgcctgga ctttggcctt 420 ttctcctgattcccagtatc tggccacagg aactcatgtc gggaaagtga acatttttgg 480 tgtggaaagtgggaaaaagg aatattcttt ggacacgaga ggaaaattca ttcttagtat 540 tgcatatagtcctgatggga aatacctagc cagtggagcc atagatggaa tcatcaatat 600 ttttgatattgcaactggaa aacttctgca taccctggaa ggccatgcca tgcccattcg 660 ctccttgaccttttccccgg actcccagct ccttgtcact gcttcagatg atggctacat 720 caagatctatgatgtacaac atgccaattt ggctggcacg ctgagcggcc atgcctcctg 780 ggtgctgaacgttgcattct gtcctgatga cactcacttt gtttccagtt cgtctgacaa 840 aagtgtaaaagtttgggatg ttggaacgag gacttgtgtt cacaccttct ttgatcacca 900 ggatcaggtctggggagtaa aatacaatgg aaatggttca aaaattgtgt ctgttggaga 960 tgaccaggaaattcacatct atgattgtcc aatttaaaca tcaaagtctc caggcttatg 1020 ctgcaaagagaatgtacgga ttgatcatga cattccttac cttcttaggc ttgtttaaaa 1080 gaaatatagcatttattgta gcaaagactt aaattttgta gatacaatat gaatcttttc 1140 atgttttattggaaatgctg ttcatacttt aacataaagc tttcttaatg caaaaagaaa 1200 aaaaaaaaaaaaaaaaaaga g 1221 3 302 PRT Homo sapiens misc_feature GenBank ID Nog1079671 3 Met Arg Lys Glu Tyr Leu Val Ser His Ile Glu Glu Asn Ala His 15 10 15 Gln Ala Asp Ile Tyr Ser Leu Asn Val Val Ala Gly Asn Leu Trp 2025 30 Ser Ala Ser Gly Asp Ser Lys Ile Lys Lys Trp Ser Ile Gly Asp 35 4045 Ala Glu His Ser Leu Val Glu Glu Ile Asp Thr Pro His Lys Leu 50 55 60Gly Val His His Leu Ala Thr Ser Leu Asp Glu Asn Val Val Val 65 70 75 SerCys Gly Phe Gly Gln Asp Val Tyr Val Trp Asn Pro Glu Thr 80 85 90 Asn GluPhe Arg Asp Leu Gly Asn Asn Ala Gln His Pro Ser Glu 95 100 105 Cys TrpSer Ser Cys Ile Ser Pro Asp Gly Gln Thr Ile Ala Phe 110 115 120 Thr SerVal Asp Gly Arg Ile Ala Val Trp Asp Asn Pro Ser Asp 125 130 135 Cys LysIle Ser Glu Leu Asp Thr Lys Gly Lys Phe Gly Leu Cys 140 145 150 Ile AspTyr Ser Pro Asn Gly Arg Phe Ile Val Ser Gly His Gln 155 160 165 Thr GlyGln Leu Phe Leu Ile Ser Thr Glu Thr Gly Arg Leu Phe 170 175 180 His ValLeu Ser Gly His Thr Ser Pro Val Arg Ser Val Ala Phe 185 190 195 Ser ProGly Ser Thr Leu Leu Ala Ala Ala Gly Asp Ser Lys Met 200 205 210 Ile ThrIle Tyr Asp Val Leu Ser Gly Asp Gln Val Gly Gln Leu 215 220 225 Arg GlyHis Ala Ala Trp Ile Phe Ala Val Ala Phe Asn Pro Val 230 235 240 Gly AspLeu Leu Leu Ser Ala Asp Val Glu Gly Lys Ile Lys Ile 245 250 255 Trp AspIle Asp Thr Met Glu Cys Ile Ser Thr Gln Ser Glu Thr 260 265 270 Asp GlyAla Ile Trp Ala Val Ala Trp Tyr Lys Asn Gly Phe Ile 275 280 285 Val AlaGly Ala Asp Lys Ser Ile Arg Trp Tyr Arg Ala Ala Ala 290 295 300 Thr Glu

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence of SEQID NO:1, b) a polypeptide comprising a naturally occurring an amino acidsequence at least 90% identical to an amino acid sequence of SEQ IDNO:1, c) a biologically active fragment of a polypeptide having an aminoacid sequence of SEQ ID NO:1, and d) an immunogenic fragment of apolypeptide having an amino acid sequence of SEQ ID NO:
 1. 2. Anisolated polypeptide of claim 1, comprising an amino acid sequence ofSEQ ID NO:1.
 3. An isolated polynucleotide encoding a polypeptide ofclaim
 1. 4. An isolated polynucleotide encoding a polypeptide of claim2.
 5. An isolated polynucleotide of claim 4, having a sequence of SEQ IDNO:2.
 6. A recombinant polynucleotide comprising a promoter sequenceoperably linked to a polynucleotide of claim
 3. 7. A cell transformedwith a recombinant polynucleotide of claim
 6. 8. A transgenic organismcomprising a recombinant polynucleotide of claim
 6. 9. A method ofproducing a polypeptide of claim 1, the method comprising: a) culturinga cell under conditions suitable for expression of the polypeptide,wherein said cell is transformed with a recombinant polynucleotide, andsaid recombinant polynucleotide comprises a promoter sequence operablylinked to a polynucleotide encoding the polypeptide of claim 1, and b)recovering the polypeptide so expressed.
 10. A method of claim 9,wherein the polypeptide comprises the amino acid sequence of SEQ IDNO:1.
 11. An isolated antibody which specifically binds to a polypeptideof claim
 1. 12. An isolated polynucleotide selected from the groupconsisting of: a) a polynucleotide comprising a polynucleotide sequenceof SEQ ID NO:2, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical to a polynucleotidesequence of SEQ ID NO:2, c) a polynucleotide complementary to apolynucleotide of a), d) a polynucleotide complementary to apolynucleotide of b) and e) an RNA equivalent of a)-d).
 13. An isolatedpolynucleotide comprising at least 60 contiguous nucleotides of apolynucleotide of claim
 12. 14. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) hybridizingthe sample with a probe comprising at least 20 contiguous nucleotidescomprising a sequence complementary to said target polynucleotide in thesample, and which probe specifically hybridizes to said targetpolynucleotide, under conditions whereby a hybridization complex isformed between said probe and said target polynucleotide or fragmentsthereof, and b) detecting the presence or absence of said hybridizationcomplex, and, optionally, if present, the amount thereof.
 15. A methodof claim 14, wherein the probe comprises at least 60 contiguousnucleotides.
 16. A method of detecting a target polynucleotide in asample, said target polynucleotide having a sequence of a polynucleotideof claim 12, the method comprising: a) amplifying said targetpolynucleotide or fragment thereof using polymerase chain reactionamplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide comprises an amino acidsequence of SEQ ID NO:1.
 19. A method for treating a disease orcondition associated with decreased expression of functional TBS,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method of screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional TBS, comprising administering to a patient in need of suchtreatment a composition of claim
 21. 23. A method of screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional TBS, comprising administering to a patientin need of such treatment a composition of claim
 24. 26. A method ofscreening for a compound that specifically binds to the polypeptide ofclaim 1, the method comprising: a) combining the polypeptide of claim 1with at least one test compound under suitable conditions, and b)detecting binding of the polypeptide of claim 1 to the test compound,thereby identifying a compound that specifically binds to thepolypeptide of claim
 1. 27. A method of screening for a compound thatmodulates the activity of the polypeptide of claim 1, said methodcomprising: a) combining the polypeptide of claim 1 with at least onetest compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method of screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a polynucleotide sequence of claim 5, themethod comprising: a) exposing a sample comprising the targetpolynucleotide to a compound, under conditions suitable for theexpression of the target polynucleotide, b) detecting altered expressionof the target polynucleotide, and c) comparing the expression of thetarget polynucleotide in the presence of varying amounts of the compoundand in the absence of the compound.
 29. A method of assessing toxicityof a test compound, the method comprising: a) treating a biologicalsample containing nucleic acids with the test compound, b) hybridizingthe nucleic acids of the treated biological sample with a probecomprising at least 20 contiguous nucleotides of a polynucleotide ofclaim 12 under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide comprising a polynucleotide sequenceof a polynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of TBS in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of TBS in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofTBS in a subject, comprising administering to said subject an effectiveamount of the composition of claim
 34. 36. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 1, themethod comprising: a) immunizing an animal with a polypeptide consistingof an amino acid sequence of SEQ ID NO:1, or an immunogenic fragmentthereof, under conditions to elicit an antibody response, b) isolatingantibodies from said animal, and c) screening the isolated antibodieswith the polypeptide, thereby identifying a polyclonal antibody whichbinds specifically to a polypeptide comprising an amino acid sequence ofSEQ ID NO:1.
 37. A polyclonal antibody produced by a method of claim 36.38. A composition comprising the polyclonal antibody of claim 37 and asuitable carrier.
 39. A method of making a monoclonal antibody with thespecificity of the antibody of claim 11, the method comprising: a)immunizing an animal with a polypeptide consisting of an amino acidsequence of SEQ ID NO:1, or an immunogenic fragment thereof, underconditions to elicit an antibody response, b) isolating antibodyproducing cells from the animal, c) fusing the antibody producing cellswith immortalized cells to form monoclonal antibody-producing hybridomacells, d) culturing the hybridoma cells, and e) isolating from theculture monoclonal antibody which binds specifically to a polypeptidecomprising an amino acid sequence of SEQ ID NO:1.
 40. A monoclonalantibody produced by a method of claim
 39. 41. A composition comprisingthe monoclonal antibody of claim 40 and a suitable carrier.
 42. Theantibody of claim 11, wherein the monoclonal antibody is produced byscreening a Fab expression library.
 43. The antibody of claim 11,wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 44. A method of detecting a polypeptidecomprising an amino acid sequence of SEQ ID NO:1 in a sample, the methodcomprising: a) incubating the antibody of claim 11 with a sample underconditions to allow specific binding of the antibody and thepolypeptide, and b) detecting specific binding, wherein specific bindingindicates the presence of a polypeptide comprising an amino acidsequence of SEQ ID NO:1 in the sample.
 45. A method of purifying apolypeptide comprising an amino acid sequence of SEQ ID NO:1 from asample, the method comprising: a) incubating the antibody of claim 11with a sample under conditions to allow specific binding of the antibodyand the polypeptide, and b) separating the antibody from the sample andobtaining the purified polypeptide comprising an amino acid sequence ofSEQ ID NO:1.
 46. A microarray wherein at least one element of themicroarray is a polynucleotide of claim
 13. 47. A method of generatingan expression profile of a sample which contains polynucleotides, themethod comprising: a) labeling the polynucleotides of the sample, b)contacting the elements of the microarray of claim 46 with the labeledpolynucleotides of the sample under conditions suitable for theformation of a hybridization complex, and c) quantifying the expressionof the polynucleotides in the sample.
 48. An array comprising differentnucleotide molecules affixed in distinct physical locations on a solidsubstrate, wherein at least one of said nucleotide molecules comprises afirst oligonucleotide or polynucleotide sequence specificallyhybridizable with at least 30 contiguous nucleotides of a targetpolynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.